Stem cells are remarkable for their ability to differentiate into diverse different tissue types, and play a central role in tissue growth, maintenance, and repair. To fulfill this role stem cells must expand their pool (during growth and development), sustain tissue homeostasis (during adulthood), and proliferate in response to injury. Well regulated mitotic divisions in stem cells ensure their remarkable ability to self-perpetuate (i.e., maintain their pool) and generate differentiated cells (i.e., replenish tissue). However, the underlying regulation is not well understood. We will investigate the roles of cellular structure and geometry in regulating stem cell division using laser microsurgery tools and time-lapse live-imaging techniques developed in our group. Several lines of evidence suggest that the orientation of the mitotic spindle is critical to stem cell divisions, and the role of the spindle in regulating stem cell divisions and differentiation will be characterized by live-imaging of spindle structures, combined with targeted perturbations using laser microsurgery to cut or remove critical structures in established model systems. The ability to selectively ablate or cut cellular structures is central to this proposal. Work in our group and others has led to the development of structural-knockout technology, whereby subcellular structures can be targeted in space and quickly ablated using tightly focused ultrashort laser pulses. Because this technique is based on highly non-linear optical breakdown (as opposed to heating or ordinary absorption) it can produce targeted sub-cellular ablations confined to regions considerably smaller than the wavelength of light; and we have demonstrated selective ablation of regions 100 nm across in cells. We will apply this technology to selectively disrupt cellular structures, including components of the cytoskeleton and mitotic spindle, to examine their role in stem cell growth and differentiation. PUBLIC HEALTH RELEVANCE: The results of proposed studies will yield a better understanding of the role of centrosomes and spindle orientation in stem cell regulation and differentiation, and through understanding these fundamental processes will greatly facilitate the development of stem cell-based therapeutics. Broad impact across the biomedical sciences will be achieved by advancing transformative laser nanosurgery technology.